1. Field of the Invention
This invention pertains to a method for the detection of viable pathogenic bacteria in a food or water sample.
2. Brief Description of the Related Art
Various pathogenic bacteria are known to be borne by food products, water or the like. These pathogenic bacteria can include Salmonella typhimurium, Listeria monocytogenes, Escherichia coli O157:H7 and the like. Salmonella typhimurium is considered to be one of the most virulent foodborne pathogens. The ingestion of these organisms via contaminated food or water may lead to salmonellosis, a serious bacterial toxi-infection-syndrome associated with gastroenteritis, typhoid and parathyphoid fevers (J. M. Jay, Foodborne gastroenteritis caused by Salmonella and Shigella. In: Modern food microbiology, 5th ed., Chapman & Hall: New York, 1996). Each year, an estimated 1.4 million human Salmonella infections occur in the United States, causing an estimated 80,000–160,000 persons to seek medical attention, and resulting in 16,000 hospitalizations and nearly 600 deaths (P. S. Mead, et al., Emerging Infect. Dis. 5 (1999) 607). Hence, detection and control of this and similar pathogens is extremely important for the safety of food products and our health. The conventional microbiological methods based on culture enrichment techniques and plating procedures are highly sensitive and selective for bacterial detection but are cumbersome and time-consuming, requiring 3–4 days for presumptive results and 5–7 days for confirmation (W. H. Andrews, et al., Food and Drug Administration Bacteriological Analytical Manual, 8th ed., AOAC International: 1995, 5.01–5.20). There is a pressing need for tests for viable pathogenic bacteria that provide results more rapidly with a sensitivity similar to, or even better than that of the conventional methods. These tests should be simple, reproducible and specific, minimizing false-positive results.
Recently, many rapid methods have been investigated to meet the need for detection of viable pathogenic bacteria in food, environmental and clinical samples. These may be broadly classified into growth methods in which cell growth is a requirement of the detection process, and non-growth methods in which no cell growth is required in the detection steps.
The antibody-based test for bacteria detection is a kind of non-growth method. Many types of immunological tests have been developed for detection of Salmonella and other pathogenic bacteria. These can be divided into those based on enzyme-linked immunosorbent assay (ELISA) (L. P. Mansfield, S. J. Forsythe, Lett. Appl. Microbiol. 31 (2000) 279; H. Van Der Zee, J. H. J. Huis In't Veld, J. AOAC international. 4 (1997) 934; K. S. Cudjoe, et al., Intl. J. Food Microbiol. 27 (1995) 11), fluorescent-antibody straining (immunofluorescent assays) (O. M. Cloak, et al., J. Appl. Microbiol. 86 (1999) 583), and various other antibody-based techniques such as immunoelectrochemical assays (J. D. Brewster, et al., Anal. Chem. 68 (1996) 4153; A. G. Gehring, et al., J. Immunol. Methods, 195 (1996) 15; Y. H. Che, et al., J. Food. Prot. 63 (2000) 1043), immunosensors (D. Ivnitski, et al., Biosens. Bioelectron. 14 (1999) 599; I. S. Park, N. Kim, Biosens. Bioelectron. 13 (1998) 1091; V. Koubova, et al., Sens. Actuators B. 74 (2001) 100), immunochromatographic methods (B. Swaminathan, Ann. Rev. Microbiol. 48 (1994) 401) and flow cytometry coupled with fluorescent antibodies (X. Wang, M. F. Slavik, J. Food Prot. 62 (1999) 717). These antibody-based methods reduce analysis time and give presumptive results in several hours to one day, when compared with culture plating procedures that require two to three days. The detection limit of these methods for Salmonella varies from 104 to 106 cells/ml. Since Salmonella cells in samples, especially, in food samples are usually present in small numbers, pre-enrichment is necessary to obtain the target cell concentration in detectable levels for applications of these methods. Furthermore, the detection sensitivity of these antibody-based methods is dominated by the quality of corresponding antibodies and labeled antibodies. Due to the low binding efficacy of Salmonella to its corresponding antibodies and the non-specific binding of antibodies, it is very difficult to further lower the detection limit for Salmonella using the antibody-based methods.
Nucleic acid-based assay for bacteria is another kind of non-growth method. The progress of a DNA amplification system called polymerase chain reaction (PCR) makes the assay much more sensitive for bacteria (W. Chen, G. Martinez, A. Mulchandani, Anal. Biochem. 280 (2000) 166). However, the PCR based assay is even more labor-intensive than ELISA and thus far more labor-intensive than modified conventional or automated methods (C. Wray, A. Wray, Salmonella in domestic animals, Oxford, N.Y., 2000). It also requires more than 4 hours to complete the test.
Impedance methods are based on the measuring the relative or absolute change in impedance, conductance and/or capacitance of a medium, as the result of metabolic activities of the microorganisms (P. Silley, S. Forsythe, J. Appl. Bacteriol. 80 (1996) 233; C. J. Felice, et al., J. Microbiol. Methods. 35 (1999) 37). Impedance enumeration of microorganisms relies on the detection time obtained from the impedance curve being inversely related to sample contamination (M. Wawerla, A. Stolle, B. Schalch, H. Eisgeruber, J. Food. Prot. 62 (1999) 1488). Impedance method has advantages over conventional microbiological methods. It reduces the analysis time to 15 h for the detection of Salmonella using selenite-cystine/trimethylamine oxide/mannitol (SCT/M) medium and had the same sensitivity as the conventional method (D. M. Gibson, J. Appl. Bacteriol. 63 (1987) 299). In fact, the impedance method is accepted by the Association of Official Analytical Chemists (AOAC) as a first action method (D. M. Gibson, P. Coombs, D. W. Pimbley, J. Assoc. Off. Anal. Chem. 75 (1992) 293).